Activated protein kinase C alpha associates with annexin VI from skeletal muscle.
We have previously detected a number of protein kinase C (PKC) alpha-binding proteins in skeletal muscle cytosol by blot overlay assay, and now identify the major, 69 kDa binding protein as annexin VI by immunoblotting and overlay assay of hydroxyapatite chromatography fractions. Annexin VI was also detected in immunoprecipitates of PKC alpha. Annexin VI and PKC alpha are both calcium-dependent phospholipid-binding proteins, and detection of the interaction was dependent on the presence of calcium and phosphatidylserine (PS). The association probably involves specific protein-protein interactions rather than mere bridging by lipid molecules: firstly, detection of PKC alpha-annexin VI complexes by overlay assay was not diminished when PS concentrations were increased over a 10-fold range, while that of other PKC alpha-binding protein complexes was reduced or abolished; secondly, the presence in the overlay assay of a PKC pseudosubstrate peptide, analogous to a PKC sequence previously found to be involved in PKC binding activity, reduced complex formation; thirdly, we were also able to detect annexin VI interaction with PKC beta by overlay of skeletal muscle cytosol, but not with PKC theta, the major novel PKC in this tissue, suggesting sequences specific to calcium-dependent PKC isoenzymes are involved. While other annexin isoforms may be PKC substrates or inhibitors, annexin VI phosphorylation by PKC alpha could not be detected after co-purification, while phosphorylation of subsequently-added histone IIIS was readily observed. Annexin VI is a major skeletal muscle protein and our data are consistent with a role for this isoform in the control of calcium-dependent PKC.
Schmitz-Peiffer
C
C
Garvan Institute of Medical Research, St. Vincent's Hospital, 384 Victoria St., Darlinghurst, Sydney, NSW 2010, Australia,
Browne
C L
CL
Walker
J H
JH
Biden
T J
TJ
D000818:Animals; D017317:Annexin A6 / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology*; D010743:Phospholipids / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011817:Rabbits; D051381:Rats
D017317:Annexin A6; D007527:Isoenzymes; D010743:Phospholipids; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D002118:Calcium